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【答案】应助回帖
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huojinlong8610: 金币+60, 翻译EPI+1, ★有帮助 2012-06-28 16:12:27
删除的单词全部用大写字母表示；添加或修改的用括号括了起来，good luck
THIS EXPERIMENT (The research) was conducted to obtain the coding sequence of Banna Mini-pig Inbred Line growth hormone (GH) gene and to STUDY (identify) its prokaryotic expression. The full-length cDNA of GH gene was cloned from pituitary by RT-PCR and inserted into pMD 18-T vector for sequencing and bioinformatics analysis. SUBCLONED (The) GH encoding mature peptide sequence (was subcloned) into the prokaryotic expressing vector pET-32α (+) directionally to constitute the recombinant plasmid pET-32α-GH. Then it (was transformed) into the E.coli Rosseta expression bacteria and induced by IPTG in different concentration and time. The recombinant expression products were detected by SDS-PAGE and confirmed by Western Blot analysis. PCR product of GH gene (contains) 690 nucletides (GenBank No. JQ177096) including a 651 bp CDS. The full-length CDS encoded 216 amino acids with a (period) of 26 amino acids of single peptide. The molecular weight of the precursor peptide was 24.4 ku and the mature peptide was 21.7 ku. Coding mature peptide sequence was comprised of 617 nucletides. It was found that the GH amino acids sequence shared 100%, 100%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 98% and 98% identity with those of WZS, Ningxiang pig, Tibetan pig, Xiang pig, Dawu pig, Taihu pig, Chenghua pig, Neijiang pig, Rongchang pig, Landrace, Yorkshire, Yanan pig, (and) Duroc, respectively. SDS-PAGE results showed that the fusion protein expressed in form of inclusion body. The optimal induction condition is 0.01 mmol•L-1 IPTG inducted for 4 (hours). Western blot analysis indicated that the recombinant protein molecular weight was (approximately) 40.6 ku and can be recognized by antiserum specifically. Collectively these results will lay the foundation for further studies of the GH effects on the dwarfism of Banna Mini-pig Inbred Line. |
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